Brief Article
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World J Gastroenterol. Sep 28, 2013; 19(36): 6044-6054
Published online Sep 28, 2013. doi: 10.3748/wjg.v19.i36.6044
In vitro effect of amoxicillin and clarithromycin on the 3’ region of cagA gene in Helicobacter pylori isolates
Javier Andrés Bustamante-Rengifo, Andrés Januer Matta, Alvaro Pazos, Luis Eduardo Bravo
Javier Andrés Bustamante-Rengifo, Andrés Januer Matta, Luis Eduardo Bravo, Registro Poblacional de Cáncer de Cali, Department of Pathology, School of Medicine, Universidad del Valle, 760043 Cali, Colombia
Alvaro Pazos, Department of Biology, Universidad de Nariño, 520002 San Juan de Pasto, Colombia
Author contributions: All the authors were involved in the acquisition and interpretation of the results, read and approved the final manuscript; Bustamante-Rengifo JA, Matta AJ and Pazos A conducted the microbiological and molecular tests; Bustamante-Rengifo JA analyzed the data; Bustamante-Rengifo JA and Bravo LE wrote, edited, and revised the manuscript.
Supported by The Administrative Department on Science and Innovation of the Republic of Colombia-COLCIENCIAS, No. RC-1106-408-20549, RC-1106-493-26237; and its program: Jóvenes Investigadores e Innovadores “Virginia Gutiérrez de Pineda”, and by the Universidad del Valle, Cali, Colombia
Correspondence to: Javier Andrés Bustamante-Rengifo, MSc, Associate Researcher, Registro Poblacional de Cáncer de Cali, Department of Pathology, School of Medicine, Universidad del Valle, Street 4 B N0. 36-00, Building 116, Floor 4°, 760043 Cali, Colombia. javierandres.bustamante@gmail.com
Telephone: +57-2-3212100-4101 Fax: +57-2-6670329
Received: January 25, 2013
Revised: April 4, 2013
Accepted: May 7, 2013
Published online: September 28, 2013
Abstract

AIM: To evaluate the in vitro effect of amoxicillin and clarithromycin on the cag pathogenicity island (cag PAI).

METHODS: One hundred and forty-nine clinical isolates of Helicobacter pylori (H. pylori) cultured from gastric biopsies from 206 Colombian patients with dyspeptic symptoms from a high-risk area for gastric cancer were included as study material. Antimicrobial susceptibility was determined by the agar dilution method. Resistant isolates at baseline and in amoxicillin and clarithromycin serial dilutions were subjected to genotyping (cagA, vacA alleles s and m), Glu-Pro-Ile-Tyr-Ala (EPIYA) polymerase chain reaction and random amplified polymorphic DNA (RAPD). Images of the RAPD amplicons were analyzed by Gel-Pro Analyzer 4.5 program. Cluster analyses was done using SPSS 15.0 statistical package, where each of the fingerprint bands were denoted as variables. Dendrograms were designed by following Ward’s clustering method and the estimation of distances between each pair of H. pylori isolates was calculated with the squared Euclidean distance.

RESULTS: Resistance rates were 4% for amoxicillin and 2.7% for clarithromycin with 2% double resistances. Genotyping evidenced a high prevalence of the genotype cagA-positive/vacA s1m1. The 3’ region of cagA gene was successfully amplified in 92.3% (12/13) of the baseline resistant isolates and in 60% (36/60) of the resistant isolates growing in antibiotic dilutions. Upon observing the distribution of the number of EPIYA repetitions in each dilution with respect to baseline isolates, it was found that in 61.5% (8/13) of the baseline isolates, a change in the number of EPIYA repetitions lowered antibiotic pressure. The gain and loss of EPIYA motifs resulted in a diversity of H. pylori subclones after bacterial adjustment to changing conditions product of antibiotic pressure. RAPD PCR evidenced the close clonal relationship between baseline isolates and isolates growing in antibiotic dilutions.

CONCLUSION: Antibiotic pressure does not induce loss of the cag pathogenicity island, but it can lead - in most cases - to genetic rearrangements within the 3’ region cagA of the founding bacteria that can affect the level of tyrosine phosphorylation impacting on its cellular effects and lead to divergence of cagA-positive subclones.

Keywords: Helicobacter pylori, Antimicrobial susceptibility, cag pathogenicity island, cagA 3’region, Random amplified polymorphic DNA-polymerase chain reaction

Core tip: This study evaluated the in vitro effect of amoxicillin and clarithromycin on the cag pathogenicity island (cag PAI). It was found that the effect of antibiotic pressure does not induce loss of cag PAI, but it can lead - in most cases - to genetic rearrangements (loss or gain of Glu-Pro-Ile-Tyr-Ala motif) within the 3’ region of cagA gene of the founding bacteria that can affect the level of tyrosine phosphorylation impacting on its cellular effects and lead to divergence of cagA-positive subclones, which as a set could alter the pathogenic process of Helicobacter pylori in cases with treatment failure.